Project Summary/Abstract There is emerging evidence that distinct populations of actively cycling and quiescent intestinal stem cells (ISCs) co-exist within the small intestine epithelium and function cooperatively in tissue renewal. Previously, we have described Lgr5 as a marker of actively proliferating ISCs that contribute to homeostatic turnover of the intestinal epithelium, whereas Bmi1 is a marker of quiescent ISCs that contribute minimally to homeostatic tissue turnover but are robustly activated for epithelial repair following tissue injury. Recently, we have discovered through transcriptional profiling of active versus quiescent ISCs that crypt Bmi1 cells isolated from Bmi1-GFP reporter mice are actually mature enteroendocrine (EE) cells. Our preliminary data suggest that there is significant cellular heterogeneity amongst FACS isolated Bmi1-GFP+ cells. This proposal explores the hypothesis that only a rare and restricted subset of Bmi1 cells marked by expression of Bmi1-GFP are EE cells that are able to reconstitute the epithelium and that these cells are most functionally active under conditions of Lgr5+ ISC loss. Furthermore, we postulate that single-cell mRNA-seq can be employed to identify such populations in an unbiased manner. Our hypothesis is evaluated by two Specific Aims: (1) to characterize the heterogeneity of Bmi1-GFP+ subsets by single-cell transcriptional profiling and (2) to isolate and functionally characterize discrete Bmi1-GFP+ subsets. Achievement of these Aims should provide insight into exciting new areas of ISC biology in which cellular plasticity of differentiated epithelial cells enables tissue reconstitution in the setting of tissue injury.